1. Field:
This disclosure is concerned generally with the production of monopclonal antibodies and specifically with a technique for the selection of desirable cell lines from a collection of human-nonhuman hybridoma cells capable of expressing monoclonal antibodies of the IgM type.
2. Prior Art:
It is well known that antibodies in general can be classified according to a standard typing scheme (i.e. IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgM, IgE, and IgA). This disclosure is especially concerned with antibodies of the IgM type.
IgM is a well known 19S immunoglobulin which comprises about 7% of the immunoglobulins found in humans. IgM antibodies have so-called J chain, light chain, and heavy chain components and are said to have an antibody valence of at least five. They are the earliest antibodies generated in an immune response. IgM antibodies tend to be very effective, especially in combating bacterial infections. They can be obtained from human plasma using known plasma fractionation techniques (e.g. U.S. Pat. No. 4,318,902 to Stephan).
The production of monoclonal antibodies, including the IgM type, has become well known since the early article by Kohler and Milstein, "Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity", Nature 256:495-497 (1975). Those antibodies can now be made in various ways such as using somatic cell hybrids (see, for example, U.S. Pat.. No. 4,172,124 to H. Koprowski et al.) or transformed cells (see, for example, U.S. Pat. No. 4,446,465 to M. Lostrom).
Purified monoclonal IgM preparations are described by D. Nau, Biochromatography, 1, No. 2, pp. 83-84 (95% pure IgM from tissue culture); M. Flashner, U.S. Pat. No. 4,604,235 (90% pure IgM from mouse ascites fluid and which was characterized as "essentially pure antibody"); J. R. Wands et al., W 082/01072 (high affinity IgM monoclonal antibodies for diagnostics, cited above); and T. Brooks et al., Amer. Lab., October, 1985 (use of hydroxylapatite for purification of mouse and human IgG and IgM).
It is well known that fusion with mouse cell lines is a common way to improve certain cell line properties. When a cell line producing human IgM is fused with a mouse cell line, mouse J chain may be incorporated in the IgM. When IgM monoclonals are expressed by such heterohydbridoma cell lines, especially if intended for therapeutic use where immunological reactions are to be avoided, it is highly desirable to develop only those cell lines expressing IgM that consists of human components for the J chain of the IgM molecule. Unfortunately, when a given IgM expressing heterohybridoma is first made (e.g., by fusion of a mouse myeloma cell and a EBV transformed human cell line), there is no practical way to assure that both the J chain and light chain components of the expressed IgM will be of or correspond to those of human origin. Whether a given cell line will express IgM having desired human components is essentially a matter of chance. Hence, it would be highly desirable to have a method available for the early screening of heterohybridoma cell lines for only human IgM components.
Quite surprisingly, we have now found that this can be done by a relatively simple technique that can be used at an early stage of cell line selection. The technique can be used with crude IgM preparations or, with non-reduced or, preferably, with reduced and further purified preparations. Clones producing desirable IgM can now be detected early in the cloning procedure, thus saving considerable time and labor. This helps avoid the costly development of undesirable cell lines so that one can focus only on those cell lines desired. Details of our methods are described below.